Reagent and test cartridge for determining clotting time

ABSTRACT

The invention is a method, reagent and test cartridge for the determination of the clotting time of a blood sample by means of a reagent containing tissue factor and a sulfatide. In an alternative embodiment, the reagent may contain tissue factor and at least one of the group consisting of a phosphatide and a sulfatide. This invention is preferably used to monitor the effectiveness of heparin therapy in patients that have been administered low to moderate heparin doses to achieve blood heparin levels from 0 to about 3 U/mL, and may also be used for determining clotting time at higher heparin levels of up to about 6 U/mL.

[0001] This application is a continuation of U.S. patent applicationSer. No. 09/645,786, filed Aug. 24, 2000, which is a non-provisional ofprovisional application serial No. 60/152,450, filed Sep. 3, 1999, bothof which are incorporated by reference as if reproduced in full below.

BACKGROUND OF THE INVENTION

[0002] The present invention relates to the field of determining theclotting time of blood samples and more specifically relates to thedetermination of the clotting time of blood samples from patientsreceiving heparin treatment, particularly patients that have beenadministered low to moderate heparin doses, as well as that of patientsthat have been administered high heparin doses.

[0003] The activated clotting time (ACT) assay is a blood test thatmonitors the effectiveness of heparin dosing. The levels of heparin thatthe ACT assay is monitoring are generally beyond the range of theactivated partial thromboplastin time (APTT) assay. Some APTT assays canmonitor plasma heparin levels as high as 1.5 U/mL (which is equivalentto a blood heparin level of about 0.75 U/mL), while the ACT assay canmonitor blood heparin levels generally as high as 6 U/mL. The higher endof the blood heparin range (high range; HR) is often used in cardiacpulmonary bypass surgery, while blood levels under 3 U/mL (moderate tolow range; LR) but above the effective range of the APTT assay, are usedin situations such as cardiac catheterization, extracorporeal membraneoxygenation (ECMO), hemodialysis, and percutaneous transluminal coronaryangioplasty (PTCA).

[0004] There are various commercially available ACT assays. Thesetypically differ in the specific component that activates clotting,which difference can affect the blood heparin range in which the assayis reliable. Consequently, these assay types are often categorized bythe heparin range and corresponding surgical or medical application. Oneexample of such a test is known as the Hemochron® sold by InternationalTechnidyne Corporation. The basic procedure for this test is as follows:A two mL sample of blood is added to a test tube containing dried celite(diatomaceous earth) and a small magnetic bar. The test tube is cappedand shaken, then placed in an instrument that starts spinning of themagnetic bar. When the blood begins to clot, the magnetic bar slows orstops spinning. The instrument then notes the length of time till themagnet stopped spinning as the celite ACT time. A variation of this testis known as the Hemochron® glass ACT assay wherein the test tube isplastic and contains glass particles with a magnetic bar and the sampleis only 0.4 mL of blood.

[0005] U.S. Pat. Nos. 4,756,884 and 5,039,617 describe an integrateddevice containing a predispensed, dry reagent in a capillary track thatcan be used to measure clotting of blood samples. The entire disclosureof these two patents is incorporated herein by reference. The assigneeof the present invention currently markets a system under thedesignation CoaguChek™ Pro and CoaguChek™ Plus. Certain challenges werepresented in designing a reagent for use in this system to measureheparin effectiveness in the range of 3 U/mL and lower. For example, theCoaguChek™ Plus/Pro system should have an assay time of 300 seconds orless, whereas traditional ACT reagents have an assay time of up to 1000seconds.

[0006] In addition, the activators that have been traditionally used inACT reagents are celite, kaolin or glass particles, which are allinsoluble particles. Such activators are typically problematic for thecartridge-reagent system employed in the CoaguChek™ Plus system whichuses the blood sample to solubilize the reagent and move the reagentwith the blood through the cartridge tracks during the coagulationactivation reaction.

SUMMARY OF THE INVENTION

[0007] Briefly stated, the invention is a method, reagent and testcartridge for the determination of the clotting time of a blood sampleby means of a reagent comprising tissue factor and a co-factor. Apreferred co-factor is a sulfatide. This invention is preferably used tomonitor the effectiveness of heparin therapy in patients that have beenadministered low to moderate heparin doses that result in blood heparinlevels from about 0 to about 3 U/mL. However, it has been surprisinglydiscovered that the invention can monitor the effectiveness of heparintherapy in patients that have been administered higher heparin dosesresulting in blood heparin levels of up to about 6 U/mL. In analternative embodiment, the sulfatide may be combined with or replacedby a phosphatide.

[0008] In accordance with the test cartridge aspect of the invention,the cartridge includes a housing containing an inlet port, a chamberunit and an exit port. The cartridge preferably further comprises afirst capillary unit for independently pumping a liquid, such as a bloodsample, from said inlet port to said chamber unit. In addition, thepreferred cartridge preferably includes a second capillary unitpositioned between and operatively connected to said chamber unit andsaid exit port for independently pumping a liquid from said chamber unitto said exit port. The inlet port, first capillary unit (if present),chamber unit, second capillary unit (if present), and exit port arepresent in a continuous capillary pathway. Contained within thecapillary pathway are a reagent comprising tissue factor and aco-factor, preferably a sulfatide. In an alternative embodiment, aphosphatide may be combined with a sulfatide or a phosphatide may be thesole co-factor.

[0009] It should be noted that, as used herein, the termsthromboplastin, tissue factor, and coagulation factor III are allintended to mean the cell-surface protein that initiates coagulation.

[0010] It should also be noted that the term sulfatide refers to a classof sulfate derivatives of cerebrosides, which have the following generalstructure:

[0011] R=fatty acid residue

[0012] The term phosphatide refers to a class of glycerol basedcompounds in which one of the hydroxyl groups is replaced with aphosphoric acid group, and the other hydroxyl groups are replaced withfatty acid esters. Phosphatides are also referred to as phospholipids,phosphoglycerides, and glycerol phosphatides. For more information onphosphatides, see Lehninger, Albert L., Biochemistry, 2^(nd) Edition,Worth Publishers, NY (1975).

[0013] The present invention, together with attendant objects andadvantages, may be better understood with reference to the detaileddescription below in connection with the attached Figures.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014]FIGS. 1A and 1B are plan and cross-sectional views of the testcartridge of the present invention.

[0015]FIG. 2 shows the basic response of heparinized samples to TF-basedreagents.

[0016]FIG. 3 shows the affect on heparin response of TF-based reagentswith varying sulfatide levels.

[0017] FIGS. 4A-4C show the graphs generated in an evaluation of sampletemperature effects on the method of the present invention.

DETAILED DESCRIPTION

[0018] The invention involves the use of tissue factor (TF) and aco-factor, preferably a sulfatide, in a method, reagent and testcartridge for determining the activated clotting time of a blood sample.In an alternative embodiment, the co-factor may be a phosphatide aloneor in combination with a sulfatide.

[0019] Tissue factor (TF), also referred to as thromboplastin andclotting factor III, is an integral membrane glycoprotein that functionsas an initiator of coagulation. TF and its properties as a biologicalinitiator of this essential hemostatic process are discussed in thereview article “Initiation of Coagulation by Tissue Factor”, by R. R.Bach, CRC Crit. Rev. Biochem. (1988) 23: 339-68.

[0020] The tissue factor component of the reagent is preferablyrecombinant human TF (rHTF). Purification of rHTF has been described inPaborsky et al., Biochemistry (1989) 28: 8072-7 and Rehemtulla et al.,Thromb. Haemost. (1991) 65: 521-7. rHTF used for the reagent of thepresent invention was purchased from Serbio (catalog No. 77800). Othersources of TF, such as natural extracts of mammalian (human, rabbit,cattle, horse, monkey, etc.) brain, lung, or platelets, etc., can alsobe used.

[0021] The basic response of heparinized samples to TF-based reagents isshown in FIG. 2. In particular, reagents were formulated as shown inTable 1, with the exception that no added sulfatide or phosphatide waspresent and the level of TF was varied from 50 ng/mL to 1000 ng/mL.These TF-based reagents were applied to the test cartridges as describedabove and samples with various heparin levels were tested in them. Athigh enough levels of tissue factor, the sensitivity of the reagent toheparin is low. As the level of tissue factor drops, sensitivityincreases, but with a loss of clot activation with samples containinghigher heparin levels.

[0022] Co-factors for TF were found that increased the heparinsensitivity in TF-based reagents. For example, sulfatides andphosphatides are co-factors that were found to control the degree ofheparin sensitivity elicited by the tissue factor. Nevertheless,phosphatides did not have the consistency in effecting tissue factorheparin sensitivity as that found for sulfatides. FIG. 3 shows theeffect on heparin response of varying sulfatide levels combined with 400ng/mL of TF. In particular, reagents were made according to Table 1below, with the exception that the TF was present at 400 ng/mL and thesulfatide concentration was varied between 0 and 0.4% of the sample,i.e., between 0 and 4 mg/mL of the sample.

[0023] Phosphatides and sulfatides can be combined at optimized ratiosand get about the same sensitivity in the assay of the present inventionas with sulfatides alone. Phosphatides are more readily available andless costly than sulfatides. Thus, in an alternative embodiment, acombination of sulfatide and phosphatide is utilized wherein the ratioof phosphatide to sulfatide is maximized yet yields optimal sensitivity.Surprisingly, ratios of phosphatide to sulfatide ranging from about 1/3to about 3/1 by weight were found to have approximately the samesensitivity as sulfatide alone. Heparin levels ranging from about 2 U/mLto about 6 U/mL can be effectively determined using an effective amountof sulfatide, phosphatide, or a combination thereof. Heparin levelsranging from 0 U/mL to about 2 U/mL can be determined using aphosphatide or a phosphatide combined with sulfatide, but it ispreferred to use only a sulfatide for this range, or phosphatidecombined with sulfatide at a ratio by weight between about 1/3 and about3/1.

[0024] A co-factor, preferably a sulfatide, and in alternativeembodiments a phosphatide alone or in combination with a sulfatide, isthus the second important ingredient in the method, reagent and testcartridge. It is to be understood that the term co-factor (or cofactor)as used herein refers to co-factors that can be utilized in combinationwith TF to determine the effectiveness of moderate to low range, andalso preferably high range, heparin dosing in accordance with thepresent invention, and achieve the desired results in less than about300 seconds. The term co-factor thus does not include insolubleparticles traditionally used as ACT activators, such as celite, kaolin,and glass particles.

[0025] Sulfatides, also known as cerebroside sulfates andsulfoglycosylspingolipids, are substances known to be present inmammalian tissues and cell membranes that show procoagulant activitythat can be attributed to contact activation reactions. Sulfatides havebeen studied as intrinsic pathway activators. For a discussion of theproperties of sulfatides and their properties as factor-XII dependentcontact activation activators, see Tans and Griffin, Blood (1982) 59-69,and Tans et al., J. Biol. Chem. (1983) 258: 8215-8222.

[0026] A preferred sulfatide for use in the present invention is bovinebrain sulfatide purchased from Life Science Research, Inc., or Sigma. Apreferred phosphatide for use in the present invention is phosphatidylcholine extracted from soybeans, available from Sigma.

[0027] The two reagent components, TF and cofactor (i.e., sulfatideand/or phosphatide), are present in appropriate amounts and proportionsin the reagent to achieve the desired results when an effective amountof the reagent is contacted with a sample containing heparin. TF isgenerally present in an amount in the reagent so that when an effectiveamount of the reagent is added to a sample (i.e., an amount sufficientto cause clotting in the desired time period), the sample comprisesbetween about 50 and about 1000 ng/mL TF, preferably between about 100and about 400 ng/mL TF, and most preferably about 100 ng/mL TF.Sulfatide or phosphatide are generally present in an amount in thereagent so that when an effective amount of the reagent is added to asample, the sample comprises at least about 1 mg/mL of cofactor, andpreferably between about 2 and about 4 mg/mL of cofactor, and mostpreferably about 3 mg/mL of cofactor. If there is a combination ofsulfatide and phosphatide, the total concentration of the combination ofcofactors present in the sample will be between 2 and 4 mg/mL, and mostpreferred about 3 mg/mL.

[0028] The cofactor (i.e., sulfatide and/or phosphatide) and TF can bepre-combined in a reagent and used wet in the method of the presentinvention. This wet reagent is aqueous and preferably provides thelevels of TF and co-factor to produce the levels of TF and co-factor inthe sample noted above.

[0029] Preferably, the TF and sulfatide and/or phosphatide are combinedin an aqueous reagent that is dried on the inside surface of a testcartridge, such as that described in U.S. Pat. No. 5,039,617. Preferredmethods of making and drying the reagent are described in this samepatent. Preferably, the resulting reagent is substantially anhydrous. Tofacilitate description, the amount of a component in an anhydrousreagent is given as the amount of the component in the aqueousformulation before drying.

[0030] In addition to the two essential components TF and a cofactor(preferably a sulfatide and/or a phosphatide), other components can bepresent in the reagent formulation. For example, bulking additives arepreferably used for ease of handling. Preferred bulking agents includesucrose and mannitol and are present at about 40 mg/mL of sample.

[0031] A buffer is also preferably included in the reagent. Glycine iscurrently preferred as the buffer and is present at about 30 mg/mL ofsample. Other suitable buffers include tris, bicine and HEPES.

[0032] A spreading agent is preferably used to facilitate coating theinside surface of the test cartridge. A preferred spreading agent isgelatin, such as porcine skin gelatin, present at about 10 mg/mL ofsample.

[0033] A surfactant, such as Triton® X-100 is preferably included atabout 0.1 mg/mL of sample. Other conventional surfactants can also beused.

[0034] Dyes are also preferably added to the formulation so that duringthe manufacture of the cartridge a quality control check can be carriedout to determine whether reagent has been added to the cartridge.Suitable dyes include Sulforhodamine B and Bromophenol blue, present atabout 0.2 mg/mL of sample.

[0035] A stabilizing agent, such as Bovine Serum Albumin (BSA) or othermammalian albumins, is preferably added at about 10 mg/mL of sample.

[0036] Because the reagent is preferably made quickly, and then dried ona cartridge that is sealed in a pouch, no preservatives are needed.Nevertheless, if the reagent is to be stored wet for any length of time,conventional preservatives can be used.

[0037] A preferred formulation for the reagent, together with a numberof variations, is set forth in Table 1. TABLE 1 ACT Reagent Formulation*Exemplary Alternative Component Preferred Composition IngredientsSulfatide Bovine Sulfatide 0.3 g/dL Phosphatide Tissue fac- Recombinanthu- 100 ng/mL Rabbit Brain TF or tor (TF) man tissue factor othermammalian (rHTF) Brain TF Bulking Ad- Sucrose 5.0 g/dL Mannitol andother ditive sugars Buffer Glycine 4.0 g/dL Lysine, Alanine, Hy-droxyproline Spreading Porcine Skin 1.0 g/dL Fish Gelatin, Calf AgentGelatin Gelatin, Collagen, Gum Hydroxypropyl Methyl Cellulose SurfactantTriton ® X-100 0.01 g/dL Tyloxapol, Pluronic L61, other Triton ® sur-factants Dye for Car- Sulforhodamine B 0.02 g/dL Bromophenol blue tridgeIn- spection Stabilizing Bovine Serum 1.0 g/dL Other mammalian al- AgentAlbumin (BSA) bumins

[0038] As noted above, in order to eliminate the handling of reagents bythe user of the device and to stabilize the reagents, the reagent ispreferably supplied within test cartridges, whereby mixing with thereagent occurs in the cartridge. The reagents may be present eitherdiffusively or non-diffusively to the surface of the cartridge, that is,adhered, absorbed, adsorbed or covalently-linked so that the reagent maybecome dissolved in the fluid or may remain fixed to the surface. Wherethe reagents are diffusively bound (non-covalently and weakly bound), avariety of situations can be accommodated. One situation is where theliquid front dissolves all of the reagent, so that the liquid frontreceives a high concentration of the reagent and most of the reactionoccurs at the liquid front. A second situation would be with an excessof a reagent of limited solubility. In this situation, the reagent maybe present in the liquid medium at a substantially uniformconcentration. A third situation is to have a deficiency of a reagent oflimited solubility, so that only the early portion of the fluid willhave a relatively constant reagent concentration. It is preferred todisperse a liquid containing the dissolved reagents onto the surface ofa reagent chamber. The liquid is spread over the chamber surface anddried under low humidity air.

[0039] In order to assure the reproducibility of distribution, varioustechniques may be employed for introducing the reagent into the chamber.Where the cartridge is produced as two parts which fit together, thereagent may be sprayed, painted, introduced into the chamber as aliquid, lyophilized or evaporated, adsorbed, covalently conjugated, orthe like. The active reagent may be combined with various stabilizers,excipients, buffers or other additives involved with the reaction.

[0040] To enhance mixing, various mechanical or ultrasonic means may beemployed to agitate the sample and reagents, where the mixing means maybe internal or external. Vibrators, ultrasonic transducers, magneticrods or other mechanical mixing means, flow disrupters, mixing bafflesor barriers, flow directors, or the like, may be employed. Theparticular manner in which agitation is provided, if provided, will varywidely depending upon the degree of agitation needed, the design of thecartridge, and the like.

[0041] The reagent need not be coated or bound to the surface of thecartridge, but may be provided as a soluble sponge or gel oralternatively, absorbed onto an insoluble sponge, membrane, paper (e.g.,filter paper) or gel which is introduced into the reaction unit. In thismanner the fluid may pass through the foam structure dissolving thereagent so as to form the reaction mixture.

[0042] The reagent may be provided in liquid form in microcapsules. Theliquid reagent could be released from the microcapsules by applyingpressure to the walls of the reaction unit, resulting in breaking of themicrocapsules and releasing the liquid reagent.

[0043] To carry out the method of the present invention, a blood sampleis brought into contact with an effective amount of the reagentdescribed above. The time until a desired or pre-determined degree ofclotting is observed in the blood sample is then measured. As notedabove, the method is preferably automated in the devices described.Alternatively, the clotting can be observed visually, or by some othertechnique and the time recorded manually.

[0044] The CoaguChek™ Pro ACT test measures both normal and prolongedactivated clotting times using fresh venous or arterial blood. TheCoaguChek™ Pro ACT test results are automatically displayed in unitsequivalent to those obtained with a commercially available ACT test.

[0045] The preferred test cartridge is shown in FIG. 1A. The devicecomprises a housing configured so that it may be introduced into aninstrument for assay determination. For example, notch 11 in housing 10is provided to allow retention of the device (e.g., by aspring-activated catch) in the instrument in which the analysis will becarried out. The housing will be constructed so as to ensure sufficientmechanical stability to withstand mechanical handling and provide forthe necessary characteristics for flow of the assay medium and detectionof the detectable signal. Entry port 20 is provided for access of ablood sample to the internal capillary of the device. A first capillarypassage 30 transports blood to reagent chamber 40 containing reagent 45.In the embodiment shown, housing 10 is provided with clear surfaces atthe location of capillary 30 in order that this section of the capillarytrack can be utilized to measure movement (and cession of movement) ofblood using a speckle-pattern detector. The blood sample, now mixed withreagent 45, exits chamber 40 and enters capillary flow unit 50, whichconnects chamber 40 to vent 60. Capillary flow unit 50 is a long,convoluted capillary pathway that provides sufficient path length forflow to be sustained for a time sufficient to measure the activatedclotting time (ACT).

[0046] A cross sectional view of the embodiment shown in FIG. 1A is setforth in FIG. 1B. This cross sectional view is taken along the lines B-Bof FIG. 1A. The construction of housing 10 from two plates, 12 and 14,is evident in this cross-sectional view. Plate 12 is essentially a flatplate that has been welded onto plate 14, which contains grooves andother depressions in its upper surface that will form the internalchambers and capillaries of the device. The two plastic pieces 12 and 14have been welded together after being properly aligned (e.g., placed inregister). “Registration” is used here in the sense of referring toproper alignment of the depressions present in the surfaces of the twopieces that are used to form the internal chambers and capillaries.Proper registration can be aided by injection molding the two pieces toprovide projections on one piece that fit into holes or depressions(other than capillary- or chamber-forming depressions) in the secondpiece.

[0047] A single convoluted depression used to form capillary channelsand chambers is present in the surface of plate 12. The cross-sectionalview shown in FIG. 1 cuts through the depression at six separatelocations, some of which (51, 52, 53, 54, and 55) are part of thecapillary flow unit 50, while the remaining location will result in theformation of the larger initiation capillary 30 and reaction chamber 40when plates 12 and 14 are welded together.

EXAMPLES

[0048] The following examples are provided by way of illustration andexplanation and as such are not to be viewed as limiting the scope ofthe present invention.

Example 1

[0049] Investigation of Sensitivity of CoaguChek™ Pro ACT Measurement toHeparin Concentration

[0050] Example 1 was carried out according to the most preferredembodiment of the present invention. In particular, Table 2 lists thelevels of the various components in the CoaguChek™ Pro ACT reagentapplied to test cartridges like those described above. The cartridgeswere produced for use with the CoaguChek™ Pro monitors described above.TABLE 2 Reagent Formulation Reagent used in first clinic Level¹ (g/dL)Component Supplier (Catalog #) 4.0 Glycine Mallinckrodt (5104) 0.01Triton X-100 Sigma (X100) 1.0 Gelatin American Gelatin Co. (low bloom)1.0 BSA Sigma (A7030) 5.0 Sucrose Sigma (S9378) 0.02 Sulfarhodamine BAldrich (23016,2) 0.30 Sulfatide 0.8% sulfatide solution 100 ng/mL rHTFSerbio (77800)

[0051] Precision studies with these cartridges on both CoaguChek™ Proand Plus monitors are shown in Table 3. Blood controls as well asheparinized blood aliquots were used in these studies. The assay resultsdisplayed by both sets of monitors were the actual times detected forclot formation. TABLE 3 Precision Study Production lot of CoaguChek ™Pro ACT cartridges A. Blood aliquots with varying heparin levels (N =18) Six monitors assaying each sample in triplicate Raw clot times BloodHeparin Pro Monitors Plus Monitors Level (U/mL) Mean S.D. C.V. Mean S.D.C.V. 0 24.7 0.7 2.8 23.5 0.6 2.5 1.5 80.9 4.0 5.0 78.8 3.3 4.1 3.0 220.323.3 10.6 246.6 30.1 12.2

[0052] B. Blood Controls (N = 18) Six monitors assaying each sample intriplicate Raw clot times Pro Monitors Plus Monitors Controls Mean S.D.C.V. Mean S.D. C.V. APTT Level 1 24.8 0.6 2.5 23.9 0.9 3.7 ACT #1 64.55.1 8.0 65.4 6.8 10.4 ACT #1 + #2 155.9 15.1 9.7 142.1 10.0 7.0

Example 2

[0053] Effect of Sample Temperature on CoaguChek™ Pro ACT Assay

[0054] An evaluation of sample temperature effects on the CoaguChek™ ProACT assay was performed and the results shown in FIGS. 4A-C. While thespecification is set at 12° to 32° C., the evaluation demonstrated noeffect on performance of the CoaguChek™ Pro ACT cartridges in atemperature range from 2° to 37° C.

Example 3

[0055] Effect of Aprotinin on CoaguChek™ Pro ACT Assay

[0056] Another study evaluated the effect of aprotinin on the CoaguChek™Pro ACT assay. While aprotinin is of greater concern for situationsrequiring the full range ACT assay (out to 6 U/mL), the known potency ofaprotinin on the conventional celite-ACT assay requires an ACT assay tobe evaluated for aprotinin affects. Table 4 contains the results of theevaluation—the aprotinin levels used in the relevant surgical proceduresare below 200 KIU/mL and this study used a level of 500 KIU/mL which issignificantly beyond the level expected in a surgical situation. TABLE 4Affect of Aprotinin on ACT Assays Samples “A” had no added aprotinin,Samples “B” had aprotinin at a level of 500 KIU/mL Blood Heparin MeanLevels (U/mL) Sample secs. 1.0 1A 53.7 1.0 1B 54.1 2.0 2A 101.7 2.0 2B109.1 3.0 3A 191.8 3.0 3B 227.2

Example 4

[0057] Effect of Hematocrit on CoaguChek™ Pro ACT Assay

[0058] Table 5 shows the results of studies to determine if hematocritvariations will affect the CoaguChek™ Pro ACT assay. The adjusted lowand high hematocrit samples gave assay results within the specificationsset with the unadjusted (normal) hematocrit samples. Therefore,hematocrit levels have not been found to influence CoaguChek™ Pro ACTresults. TABLE 5 A. CoaguChek ™ Plus hematocrit study with CoaguChek ™Pro ACT (R&D lot) Low and high hematocrit heparinized blood samplesassayed in duplicate; unadjusted hematocrit (normal) heparinized bloodsamples assayed in quadru- plicate Specification: 80% to 120% of SampleMean Nor- (HCT) mean seconds mal Donor #1 Low (25%) 86.8 (1.5 U/mL)Normal (44%) 93.4 74.7 to 112.0 High (56%) 107.6 Donor #2 Low (21%) 86.6(2.0 U/mL) Normal (42%) 88.0 70.4 to 105.8 High (54%) 88.1 Donor #3 Low(21.5%) 72.0 (2.0 U/mL) Normal (44%) 85.4 68.3 to 102.5 High (55%) 90.6Donor #4 Low (23.5%) 63.3 (1.5 U/mL) Normal (46%) 71.3 57.0 to 85.6 High(63%) 82.2 Donor #5 Low (22%) 88.4 (2.0 U/mL) Normal (41%) 105.9 84.7 to127.1 High (55%) 122.6

[0059] TABLE 5 B. CoaguChek ™ Pro hematocrit study with CoaguChek ™ ProACT (Production lot ACT021898 All samples assayed on three monitorsthree times Blood heparin level adjusted to 1.5 U/mL Specification: 80%to 120% of Low Normal High Mean Normal Mean: 88.6 84.5 100.3 67.6 to101.4 S.D.: 6.6 1.3 5.5 C.V.: 7.5 1.6 5.4 Hematocrit: 21% 43.5% 55%

Example 5 Testing of Assay Sensitivity at Higher Dosing Levels

[0060] The protocol of Example 1 was followed, except that samplescontaining up to 6 U/mL heparin and higher were tested. It wassurprisingly discovered that, in addition to its usefulness fordetermining the effectiveness of low to moderate heparin dosing, thepresent invention may also be used to determine the effectiveness of HRheparin dosing up to about 6 U/mL.

Example 6

[0061] Testing of Alternative Co-Factor Compositions

[0062] The protocol of Example 1 was followed, except that a phosphatidewas used in place of a sulfatide, or mixed with a sulfatide. Thecombined weight of the sulfatide and the phosphatide was maintained atthe same level as if the sulfatide was present alone (i.e., an equalweight of the sulfatide was subtracted for the amount of the phosphatideadded). Varying ratios by weight of the phosphatide to the sulfatidewere utilized in reagent compositions with TF. At moderate to highheparin levels of about 2 U/mL up to about 6 U/mL, compositionscontaining a phosphatide in place of a sulfatide, or a combination of asulfatide and a phosphatide were found to have about equal effectivenessand sensitivity. For low range heparin dosing of about 0 to about 2U/mL, it is preferred to use either a sulfatide alone, or a phosphatideand a sulfatide combination at between about 1/3 to about 3/1 ratio byweight.

[0063] While preferred and exemplary embodiments of the presentinvention have been described, the present invention may be practicedother than as specifically described herein and still fall within thescope of the present invention as set forth in the following claims.

1. A reagent for the determination of the clotting time of a bloodsample from a patient receiving heparin treatment, wherein the clottingtime is used to determine the effectiveness of the treatment, comprisingtissue factor and a sulfatide in relative amounts sufficient todetermine the effectiveness of heparin treatment in relation to clottingtime in a sample from a patient receiving sufficient heparin to have ablood heparin level of up to about 6 U/mL.
 2. The reagent of claim 1,wherein said reagent is anhydrous.
 3. The reagent of claim 1, whereinsaid tissue factor is present in sufficient quantity in said reagent sothat when an effective amount of said reagent is added to a blood sampleto clot the sample, the sample comprises between about 50 and about 1000ng/mL tissue factor.
 4. The reagent of claim 3, wherein a sample aftercontact with an effective amount of said reagent comprises about 100ng/mL tissue factor.
 5. The reagent of claim 1, wherein said sulfatideis present in sufficient quantity in said reagent so that when aneffective amount of said reagent is added to a blood sample to clot thesample, the sample comprises between about 1 and about 4 mg/mLsulfatide.
 6. The reagent of claim 5, wherein the sample after contactwith an effective amount of said reagent comprises about 3 mg/mLsulfatide.
 7. The reagent of claim 1, wherein said reagent furthercomprises a buffer and a stabilizer.
 8. A test cartridge for thedetermination of the clotting time of a blood sample from a patientreceiving heparin treatment, wherein the clotting time is used todetermine the effectiveness of the treatment, comprising: a housingcontaining an inlet port, a chamber unit, and an exit port, said inletport, chamber unit, and exit port being present in a continuouscapillary pathway; and a reagent in said capillary pathway comprisingtissue factor and a sulfatide
 9. The test cartridge according to claim8, wherein said tissue factor is recombinant human tissue factor andsaid sulfatide is bovine brain sulfatide.
 10. The test cartridge ofclaim 8, wherein said tissue factor is present in sufficient quantity insaid reagent so that when an effective amount of said reagent iscontacted with a blood sample to clot the sample, the sample comprisesbetween about 50 and about 1000 ng/mL tissue factor
 11. The testcartridge of claim 10, wherein a sample after contact with an effectiveamount of said reagent comprises about 100 ng/mL tissue factor.
 12. Thetest cartridge of claim 8, wherein said sulfatide is present insufficient quantity in said reagent so that when an effective amount ofsaid reagent is contacted with a blood sample to clot the sample, thesample comprises between about 1 and about 4 mg/mL sulfatide.
 13. Thetest cartridge of claim 12, wherein the sample after contact with aneffective amount of said reagent comprises about 3 mg/mL sulfatide. 14.The test cartridge according to claim 8, wherein said reagent furthercomprises a buffer and a stabilizer.
 15. A test cartridge for thedetermination of the clotting time of a blood sample from a patientreceiving heparin treatment, wherein the clotting time is used todetermine the effectiveness of the treatment, comprising: a housingcontaining an inlet port, a chamber unit, an exit port, a firstcapillary unit for independently pumping a liquid from said inlet portto said chamber unit, and a second capillary unit positioned between andoperatively connected to said chamber unit and said exit port forindependently pumping a liquid from said chamber unit to said exit port;wherein said inlet port, first capillary unit, chamber unit, secondcapillary unit, and exit port are present in a continuous capillarypathway; and a reagent in said capillary pathway comprising tissuefactor and a sulfatide.
 16. A reagent for the determination of theeffectiveness of heparin treatment in a patient receiving same,comprising tissue factor and at least one co-factor selected from thegroup consisting of a phosphatide and a sulfatide, wherein when asufficient quantity of said reagent is contacted with a blood samplefrom a patient, clotting time can be used to determine to theeffectiveness of heparin therapy to the patient.
 17. The reagent ofclaim 16, wherein said reagent is anhydrous.
 18. The reagent of claim16, wherein said tissue factor is present in sufficient quantity in saidreagent so that when an effective amount of said reagent is added to ablood sample to clot the sample, the sample comprises between about 50and about 1000 ng/mL tissue factor.
 19. The reagent of claim 18, whereinthe sample after contact with an effective amount of said reagentcomprises about 100 ng/mL tissue factor.
 20. The reagent of claim 16,wherein said at least one co-factor selected from the group consistingof a phosphatide and a sulfatide is present in sufficient quantity insaid reagent so that when an effective amount of said reagent is addedto a blood sample to clot the sample, the sample comprises a combinedtotal of said phosphatide and said sulfatide combined between about 1and about 4 mg/mL.
 21. The reagent of claim 20, wherein the sample aftercontact with an effective amount of said reagent comprises a combinedtotal of said phosphatide and said sulfatide of about 3 mg/mL.
 22. Thereagent of claim 16, wherein said reagent further comprises a buffer anda stabilizer.
 23. A test cartridge for the determination of theeffectiveness of heparin treatment in a patient receiving same,comprising: a housing containing an inlet port, a chamber unit, and anexit port, said inlet port, chamber unit, and exit port being present ina continuous capillary pathway; and a reagent in said capillary pathwaycomprising tissue factor and at least one co-factor selected from thegroup consisting of a phosphatide and a sulfatide.
 24. The testcartridge according to claim 23, wherein said tissue factor isrecombinant human tissue factor, said sulfatide is bovine brainsulfatide, and said phosphatide is phosphatidyl choline.
 25. The testcartridge of claim 23, wherein said tissue factor is present insufficient quantity in said reagent so that when an effective amount ofsaid reagent is contacted with a blood sample to clot the sample, thesample comprises between about 50 and about 1000 ng/mL tissue factor.26. The test cartridge of claim 25, wherein the sample after contactwith an effective amount of said reagent comprises about 100 ng/mLtissue factor.
 27. The test cartridge of claim 23, wherein at least oneco-factor from said group consisting of a phosphatide and a sulfatide ispresent in sufficient quantity in said reagent so that when an effectiveamount of said reagent is contacted with a blood sample to clot thesample, the sample comprises a combined total of said phosphatide andsaid sulfatide between about 1 and about 4 mg/mL.
 28. The test cartridgeof claim 27, wherein the sample after contact with an effective amountof said reagent comprises a combined total of said phosphatide and saidsulfatide of about 3 mg/mL.
 29. The test cartridge according to claim27, wherein said reagent further comprises a buffer and a stabilizer.30. A reagent for use in determining the effectiveness of heparintreatment in patients receiving same, comprising a sulfatide and aphosphatide, wherein said sulfatide and said phosphatide are present ina ratio by weight of said phosphatide to said sulfatide of about 1/3 toabout 3/1, and said reagent can determine heparin treatmenteffectiveness in patients receiving sufficient heparin to have bloodheparin levels between about 0 U/mL and about 6 U/mL.
 31. The reagent ofclaim 30, further comprising tissue factor.
 32. A test cartridge for thedetermination of the effectiveness of heparin treatment in patientsreceiving same, comprising: a housing containing an inlet port, achamber unit, an exit port, a first capillary unit for independentlypumping a liquid from said inlet port to said chamber unit, and a secondcapillary unit positioned between and operatively connected to saidchamber unit and said exit port for independently pumping a liquid fromsaid chamber unit to said exit port; wherein said inlet port, firstcapillary unit, chamber unit, second capillary unit, and exit port arepresent in a continuous capillary pathway; and a reagent in saidcapillary pathway comprising tissue factor and a phosphatide.
 33. Areagent for use in determining the effectiveness of heparin treatment inpatients receiving sufficient heparin to have a blood heparin levelbetween about 0 U/mL and about 6 U/mL, comprising tissue factor and acofactor, wherein, when an effective amount of said reagent is contactedwith a blood sample from a patient having a blood heparin level betweenabout 0 U/mL and about 6 U/mL, a predetermined degree of clotting isreached in less than about 300 seconds.
 34. The reagent of claim 33,wherein said cofactor comprises at least one of the group consisting ofa sulfatide and a phosphatide.